Mir-340-FHL2 Axis Inhibits Cell Growth and Metastasis in Ovarian

Huang et al. Cell Death and Disease (2019) 10:372 https://doi.org/10.1038/s41419-019-1604-3 Cell Death & Disease

ARTICLE Open Access miR-340-FHL2 axis inhibits cell growth and metastasis in ovarian cancer Zheng Huang1,QiuxiaLi1, Kaili Luo1, Qinkai Zhang1,JingwenGeng1, Xunzhu Zhou1, Yesha Xu1,MengyaoQian1, Jian-an Zhang2, Liying Ji1 and Jianmin Wu1

Abstract Although increasing evidence indicated that deregulation of microRNAs (miRNAs) contributed to tumor initiation and progression, but little is known about the biological role of miR-340 in ovarian cancer (OC). In this study, we found that miR-340 expression was downregulated in OC tissues compared with its expression in normal ovarian epithelium and endometrium, and treatment with 5-aza-2′-deoxycytidine (5-Aza-dC) or trichostatin A (TSA) increased miR-340 expression in OC cells. In addition, ectopic miR-340 expression inhibited OC cell growth and metastasis in vitro and in vivo. Four and a half LIM domains protein 2 (FHL2) was conﬁrmed as a direct target of miR-340 and silencing FHL2 mimicked the effects of miR-340 in OC cells. Further mechanistic study showed that miR-340 inhibited the Wnt/ β-catenin pathway by targeting FHL2, as well as downstream cell cycle and epithelial-to-mesenchymal transition (EMT) signals in OC cells. Moreover, the greatest association between miR-340 and FHL2 was found in 481 ovarian serous cystadenocarcinoma tissues via pan-cancer analysis. Finally, we revealed that lower miR-340 or higher FHL2 was associated with poor OC patient outcomes. Our ﬁndings indicate that the miR-340-FHL2 axis regulates Wnt/β-catenin signaling and is involved in tumorigenesis in OC. Therefore, manipulating the expression of miR-340 or its target genes is a potential strategy in OC therapy. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,;

– Introduction leads to a poor 5-year survival rate5 7. Thus, increased Human ovarian cancer (OC) is the leading cause of understanding of the mechanisms driving OC and iden- – death from gynecologic malignancies1 3. Worldwide, tiﬁcation of new therapeutic targets are needed to ~295,400 new cases of OC have been diagnosed and improve OC treatments. 184,800 women are expected to succumb to the disease in Wingless/integrated (Wnt)-mediated signal transduc- 20184. This case-to-fatality ratio is nearly twice that of tion pathways are known to regulate development and – breast cancer4. Due to high tumor heterogeneity and lack stemness, but are also tightly associated with cancer8 10. of reliable detection markers, the majority of OC patients Activated Wnt signaling has been reported to mediate OC are diagnosed at advanced stages. Despite high response initiation and progression by regulating cell proliferation rates for initial chemotherapy, many patients develop and apoptosis, even epithelial-to-mesenchymal transition recurrent disease with chemoresistance which ultimately (EMT)11,12. The Wnt signaling pathway is commonly divided into β-catenin-dependent (canonical) and β-catenin-independent (noncanonical) signaling8,9. The Correspondence: Jianmin Wu ([email protected])([email protected]. canonical pathway is activated upon Wnt ligand binding cn) to Frizzled receptors and LDL-receptor-related protein 1Institute of Genomic Medicine, Wenzhou Medical University, 325000 Wenzhou, Zhejiang, P. R. China (LRP) co-receptors. These interactions disrupt the ade- 2Department of Obstetrics and Gynecology, The Second Afﬁliated Hospital and nomatous polyposis coli (APC)/Axin/casein kinase 1 Yuying Children of Wenzhou Medical University, 325027 Wenzhou, Zhejiang, alpha (CK1A)/glycogen synthase kinase 3 beta (GSK3β) P. R. China These authors contributed equally: Zheng Huang, Qiuxia Li, Kaili Luo complex, which results in the translocation and Edited by A. Peschiaroli

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accumulation of β-catenin in the nucleus, where it acti- (DMEM, BI, Israel). HEK293T and SKOV3 cells were vates target gene expression by interacting with T-cell grown in Roswell Park Memorial Institute (RPMI) 1640 factor/lymphoid enhancer-binding factor (TCF/LEF) medium (BI). All media were supplemented with 10% fetal transcription factors8,9. bovine serum and the cells were incubated at 37 °C in a Four and a half LIM domains protein 2 (FHL2) is a humidified incubator with 5% CO2. Cell line authentica- multifunctional scaffolding protein regulating signaling tion was achieved by genetic profiling using polymorphic cascades and gene transcription13. FHL2 has been impli- short tandem repeat loci. The cells were treated with 5- cated in the Wnt/β-catenin signaling pathway14. FHL2 aza-2′-deoxycytidine (5-Aza-dC) or trichostatin A (TSA) was first reported as a coactivator of β-catenin15 that at the indicated concentrations for 48 or 24 h, respec- promoted differentiation of mouse myoblasts16.In tively, before subsequent analysis. osteosarcoma and colon cancer, FHL2 can stabilize nuclear β-catenin, resulting in enhanced β-catenin Microarray data analysis transactivation activity17,18. FHL2 was also shown to Microarray datasets were obtained from Gene Expres- promote tubular EMT through modulation of β-catenin sion Omnibus (GEO): GSE71477 (eight groups including signaling in fibrotic kidneys19. Consistent with aberrant 19 paired tissue samples representing OC and endome- Wnt/β-catenin signaling, the expression of FHL2 was trium) and GEO: GSE47841 (including 9 ovarian surface dysregulated in several types of cancers, including epithelium scraping samples and 12 high-grade serous – OC18,20 22. Recently, FHL2 has been shown to regulate ovarian carcinoma samples). miRNAs upregulated by ovarian granulosa cell tumor progression via controlling >1.25-fold or downregulated by <0.8-fold (P < 0.025) in AKT1 transcription23. Nevertheless, the precise molecular the OC samples were considered to be dysregulated. The mechanism of FHL2’s involvement in OC remains far heat-map was plotted using MEV 4.9.0 software. from being understood. MicroRNAs (miRNAs) are non-coding RNAs ~22 Establishment of stable OC cell lines nucleotides in length that can influence a wide range of To generate stable miR-340-overexpressing cells, A2780 biological processes by repressing target mRNA levels24,25. and SKOV3 cells were transduced with a lentivirus Increasing evidence has demonstrated that miRNAs are expressing miR-340 or a negative control (Genechem, highly dysregulated in cancers, suggesting that they can Shanghai, China), according to the manufacturer’s – function as therapeutic targets in tumorigenesis24 26. instructions, and selected using 500 ng/mL puromycin. MicroRNA-340 (miR-340) has been identified as one of After 6 days, puromycin-resistant cell pools with green the miRNAs that are downregulated in several types of fluorescent protein signals were identified using laser cancer. miR-340 can inhibit cell proliferation, induce cell scanning confocal microscopy, collected, and then verified apoptosis, and reduce cell migration and invasion by tar- by real-time RT-PCR. To generate stable miR-340 and geting multiple oncogenes in breast cancer, gastric cancer, FHL2 co-expressing cells, the lentiviral particles were first – glioblastoma, and non-small cell lung cancer27 32. produced in HEK293T cells co-transfected with p-FHL2 Accordingly, some signaling pathways related to tumor- plasmid (EX-M0686-Lv105-5, Genecopoeia, USA) or igenesis, such as Janus kinase/signal transducers and control plasmid (EX-eGFP-Lv105, Genecopoeia) along activators of transcription (JAK/STAT), Wnt/β-catenin, with psPAX2 (Addgene, Watertown, MA, USA) and and RAS/RAF/mitogen-activated protein kinase (MAPK), pMD2.G (Addgene) plasmids. Lv-miR-340-SKOV3 cells – were shown to be suppressed by miR-34033 35. were then transduced with the resulting lentiviral particles The purpose of the present study was to investigate the and selected using 1000 ng/mL puromycin. After 6 days, molecular function of miR-340, as well as its regulatory puromycin-resistant cell pools were collected and verified mechanisms in OC. We found that downregulation of miR- by western blotting. 340 in OC was due to the promoter hypermethylation, which is associated with poor patient outcomes. miR-340 siRNA and transfection inhibited cell growth, promoted apoptosis, and reduced siRNAs targeting FHL2 were obtained from Gene- migratory and invasive ability in vitro and in vivo by directly Pharma (Shanghai, China). siRNA was transfected at a targeting FHL2. Moreover, we identified that the miR-340- final concentration of 50 nM using Lipofectamine 2000 FHL2 axis regulated Wnt/β-catenin signaling in OC cells. (Life Technologies) according to the manufacturer’s instructions. The cells were collected 48 h after transfec- Methods tion for subsequent analysis. Cell culture and drug treatment The human A2780, SKOV3, and HEK293T cell lines Real-time RT-PCR were acquired from ATCC (Manassas, VA, USA). A2780 Total RNA was isolated using TRIzol (Thermo Fisher cells were grown in Dulbecco’s modified Eagle’s medium Scientific) and treated with RNase-free DNase I (Promega,

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Madison, WI, USA) for 30 min. To quantify mRNA levels, 100 for 30 min at 4 °C, the cells were washed and analyzed the DNA-free RNA was reverse-transcribed using the M- by flow cytometry (C6, BD, NJ, USA) to detect the DNA MLV Reverse Transcription Kit (Promega) according to content of the stained cells. For cell apoptosis analysis, the the manufacturer’s instructions. Real-time PCR (qPCR) cells were stained with the PE Annexin V Apoptosis ® was performed using SYBR Premix Ex Taq (TaKaRa). Detection Kit (#559763, BD, USA) for 15 min at room Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) temperature, following the manufacturer’s instructions. mRNA expression levels served as a reference control. Flow cytometry was then performed to determine the The sequences of the qPCR primers are listed in Sup- percentage of apoptotic cells. plementary Table S1. To evaluate miR-340 expression, reverse transcription and qPCR were performed using the Immunofluorescence staining bulge-loop miRNA qPCR primer set (RiboBio, Guangz- Immunofluorescence assays were performed as descri- hou, China) according to the manufacturer’s instructions, bed previously36. The primary antibody, anti-Ki67 (sc- and the data were normalized to the expression levels of 23900), was obtained from Santa Cruz Biotechnology human small nuclear RNA U6. (Santa Cruz, CA, USA). Anti-FHL2 (ab12327) was obtained from Abcam (Cambridge, UK). Anti-β-catenin Dual-luciferase reporter assay (#8480) was obtained from Cell Signaling Technology The FHL2 3′-untranslated region (UTR) reporter plas- (Danvers, MA, USA). The fluorescein isothiocyanate mid (Wt-FHL2 3′UTR) was generated by cloning the full (FITC)-conjugated donkey anti-mouse (D110081-0100) length of FHL2 3′-UTR sequences (NM_001039492) into and Cy3-conjugated donkey anti-rabbit (D110052-010) the pGL3-control vector (Promega) at the Xba1 site. To secondary antibodies were obtained from Sangon Biotech generate the mutant FHL2 reporter (Mut-FHL2 3′UTR), (Shanghai, China). the seed region of the FHL2 3′-UTR was mutated using the QuickMutation™ Site-Directed Mutagenesis Kit 5-Ethynyl-2′-deoxyuridine (EdU) proliferation assay (Beyotime, Shanghai, China). HEK293T or SKOV3 cells Logarithmically proliferating Lv-miR-340-A2780 or Lv- were seeded in 96-well plates and co-transfected with miR-340-SKOV3 cells were seeded in 96-well plates (8 × 100 ng of the firefly luciferase reporter vectors, Wt-FHL2 104 cells/well) 12 h before staining with the Cell-Light™ ® 3′UTR or Mut-FHL2 3′UTR, and 10 ng Renilla luciferase EdU Apollo 643 In Vitro Imaging Kit (RiboBio) according control vector (pRL-CMV), with 5 pmol miRNAs (Ribo- to the manufacturer’s protocol. Briefly, the cells were Bio), using Lipofectamine 2000. Luciferase activities were incubated with 50 μM EdU for 2 h before fixation with 4% measured 48 h after transfection using the Dual-Glo paraformaldehyde, permeabilization with 0.5% Triton X- Luciferase Assay System (Promega), in which firefly luci- 100, and EdU staining. The cell nuclei were stained with ferase activity was normalized to Renilla luciferase activity. Hoechst 33342 for 30 min. The number of EdU-positive cells in five random fields was counted under laser scan- Cell viability and colony formation assay ning confocal microscopy. Cell proliferation/viability was determined as described ® previously36, using the CellTiter 96 AQueous One In vitro migration and invasion assays Solution Cell Proliferation Assay Kit (3-(4,5-dimethyl- The migration and invasion assays were conducted as thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe- described previously37;5×104 cells were used for nyl)-2H-tetrazolium (MTS) assay, Promega) according to migration (SKOV3 for 6 h and A2780 for 12 h) and the manufacturer’s instructions. For the colony formation invasion (SKOV3 for 18 h and A2780 for 24 h). assay, treated cells were seeded in six-well plates at a density of 500 cells per well and cultured for 14 days. The Wound-healing assay colonies were then fixed with cold methanol and stained For the wound-healing assay, the cells were seeded in with 0.1% crystal violet; colonies comprising more than 50 six-well plates at a number that reached confluency after cells were counted. an overnight incubation. A horizontal line scratch was created in the cell monolayer using a pipette tip assisted Cell cycle and apoptosis analysis with a ruler. Then, the cells were lightly washed with PBS The treated cells were harvested at 80% confluence and twice and incubated in serum-free medium. The scratch washed with ice-cold phosphate-buffered saline (PBS) healing ability was recorded by taking pictures 0, 24, and twice. For cell cycle analysis, the cells were fixed with cold 48 h after scratching. 70% ethanol at 4 °C overnight, washed with ice-cold PBS twice, and then filtered with a 0.05-mm cell strainer. After Western blot incubation with PBS containing 50 μg/mL propidium Western blot analysis was performed as described pre- iodide (PI), 100 μg/mL RNase A, and 0.2% (v/v) Triton X- viously36. The primary antibodies were as follows: anti-

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p27 (ab193379, Abcam), anti-phosphorylated Rb (Ser795) The Cancer Genome Atlas (TCGA) data set analysis (#9301, Cell Signaling Technology), anti-E2F1 (#3742, TCGA gene expression data and clinical data were Cell Signaling Technology), anti-p21 (#2947, Cell Signal- obtained from TCGA Data Portal (http://cancergenome. ing Technology), anti-caspase-3 antibody (ab32351, Epi- nih.gov/). miRNA and mRNA expression was determined tomics), anti-FHL2 (ab12327, Abcam), anti-β-catenin using the HiSeq™ 2000 platform. Reads per million (RPM) (#8480, Cell Signaling Technology), anti-phosphorylated was used to quantify miR-340 expression levels from the β-catenin (Ser33/37/Thr41) (#9561, Cell Signaling Tech- miRNA-Seq datasets. FHL2 expression was calculated as nology), anti-cyclin D1 (60186-lg, Proteintech), anti-p53 reads per kilobase per million mapped reads (RPKM) (10442-1-AP, Proteintech), anti-PUMA (ab33906, Epi- values. The normalized values of miR-340 and FHL2 tomics), anti-E-cadherin (20874-1-AP, Proteintech), anti- expression were converted to log2-transformed values. N-cadherin (22018-1-AP, Proteintech), and anti-GAPDH The relationship between miR-340 expression and survi- (#2118, Cell Signaling Technology). val was explored by separating the cases into two groups by the data-driven approach. Animal studies For the xenograft studies, 6-week-old female BALB/c Statistical analysis nude mice were purchased from SLAC Laboratory Animal Data are presented as means ± SD or ±SEM. Unless noted Co., Ltd (Shanghai, China) and maintained under otherwise, each experiment was carried out in triplicate. pathogen-free conditions. A total of 4.5 × 106 Lv-miR-340- Statistical significance was determined by two-tailed Stu- A2780 or control cells in 100 µL PBS were subcutaneously dent’s t-test or Mann–Whitney U-test. Differences between (s.c.) injected into the rear flank of the mice. Tumors were groups were determined using two-way analysis of variance measured using a caliper, and tumor volume was calcu- (ANOVA). P<0.05 was considered statistically significant. lated using the following formula: V = L × W2 × 0.5236 (L = long axis, W = short axis). For in vivo metastasis assays, Results 1×106 Lv-miR-340-SKOV3 or 2 × 106 Lv-miR-340-A2780 miR-340 is downregulated in human OC cells and the corresponding control cells in 200 µL PBS To identify novel miRNAs involved in OC tumorigen- were injected intraperitoneally (i.p.) into the nude mice. esis, we analyzed two datasets from GEO (GSE71477, After 70 days, the mice were photographed, and then 1155 miRNAs and GSE47841, 914 miRNAs), which con- euthanized. The nodules throughout the peritoneal cavity tains most known miRNAs, and 36 (15 downregulated were counted and the ascites weight was measured. The and 21 upregulated) common significantly dysregulated intestines were subjected to fluorescent image detection miRNAs were identified (P < 0.025 and fold-change (FC) using the IVIS Lumina III In Vivo Imaging System (Per- <0.8 or >1.25) (Fig. 1a–c). Consistent with previous – kinElmer, MA, USA). All animals were maintained and reports38 42, this list contained miR-145, miR-140, the treated in accordance with the guidelines of the Institu- miR-200 family, and miR-20a, suggesting its reliability tional Animal Care and Use Committee of Wenzhou (Supplementary Table S2, Fig. 1b). As one of the down- Medical University. regulated miRNAs, the levels of miR-340 among 30 types of cancers were lowest in OC (Supplementary Fig. S1A), Immunohistochemistry so it was selected for further in-depth mechanistic study. Paraffin sections (5 μm) from tumor tissue samples were Previous studies have shown that the downregulated deparaffinized in 100% xylene and rehydrated with a miRNAs are associated with DNA hypermethylation in decreasing ethanol series and then water, according to their promoters43. Thus, we first analyzed the 2-kb region standard protocols. Heat-induced antigen retrieval was upstream of miR-340 by EMBOSS44, and three putative performed in 0.01 mol/L sodium citrate buffer (pH 6.0) CpG islands (Observed/Expected ratio (Obs/Exp ratio) for 10 min at 100 °C. Endogenous peroxidase activity and >0.60, C+G>50% and length >200) were found (Supple- non-specific antigens were blocked, followed by incuba- mentary Fig. S1B), which could be involved in the tion with anti-FHL2 antibody (ab12327, Abcam), anti- downregulation of miR-340 in OC cells. As expected, Ki67 antibody (sc-23900, Santa Cruz), or anti-caspase-3 treatment with 5-Aza-dC or TSA restored the levels of antibody (ab32351, Epitomics), overnight at 4 °C. After miR-340 in A2780 and SKOV3 cells (Fig. 1d, e), sug- washing, the sections were incubated with Poly-HRP- gesting that downregulation of miR-340 is due to its Conjugated Anti-Rabbit IgG or Poly-HRP-Conjugated promoter hypermethylation in OC cells. Anti-Mouse IgG (Sangon Biotech) for 45 min at room temperature. The sections were visualized with 3,3′-dia- miR-340 inhibits growth of OC cells and promotes minobenzidine (DAB), counterstained with hematoxylin, apoptosis in vitro and in vivo mounted in neutral gum and analyzed using a bright field To further explore the biological role of miR-340 in OC, microscope. A2780 and SKOV3 cells were transduced with miR-340-

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Fig. 1 miR-340 is downregulated in human ovarian cancer (OC). a Dysregulated miRNAs in OC compared with the corresponding control in both GSE71477 and GSE47841 datasets, in which 15 are downregulated and 21 are upregulated (P < 0.025, fold change <0.8 or >1.25). b Heatmap showing the hierarchical clustering of miRNAs expressed in human OC from GSE71477 dataset. c Expression of miR-340 was signiﬁcantly downregulated in OC compared with that of ovarian epithelium (GSE47841) or endometrium (GSE71477). Data are shown as means ± SEM. d, e A2780 and SKOV3 cells were treated with 5-aza-2′-deoxycytidine (5-Aza-dC) (d) for 48 h or trichostatin A (TSA) (e) for 24 h. The relative expression of miR-340 was determined by real-time RT-PCR. Data are shown as means ± SD from three independent experiments. *P < 0.05, **P < 0.01 by Student’s t-tests

overexpressing lentivirus (Lv-miR-340) or control lenti- corresponding Lv-miR-NC group (Supplementary virus (Lv-miR-NC). More than 300-fold increase in miR- Fig. S2). Subsequently, OC cell proliferation was assessed 340 expression was observed in Lv-miR-340-A2780 and in vitro. Overexpression of miR-340 resulted in a sig- Lv-miR-340-SKOV3 cells compared with that of the niﬁcant decrease in cell viability compared with that in the

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miR-NC group by MTS assay, both in A2780 and SKOV3 Lv-miR-340 cells showed smaller ascites volumes cells (Fig. 2a). Ectopic miR-340 expression blocked pro- (Fig. 4d), although there was no significant difference in liferation of OC cells in the colony formation assay ascites weight between the two groups (Supplementary (Fig. 2b). Cell-cycle analysis revealed that miR-340 over- Fig. S3B). A few small nodules throughout the peritoneal expression significantly arrested both A2780 and SKOV3 cavity were also detected in the Lv-miR-340 group cells at the G1-S transition, as indicated by a marked (Fig. 4e). Fluorescence imaging revealed that the mice accumulation of cells in the G1 peak and a reduction of inoculated with Lv-miR-340 cells had weaker green fluor- cells in S phase (Fig. 2c). Decreased expression of Ki67 (a escence signals on their mesentery than did mice injected cell proliferation marker) indicated that miR-340 sig- with Lv-miR-NC cells (Fig. 4f). These results demonstrate nificantly inhibited OC cell proliferation (Fig. 2d). Fur- that miR-340 is an OC metastatic inhibitor in vivo. thermore, an EdU incorporation assay for detecting DNA replication showed that the cell proliferation of A2780 FHL2 is a direct target of miR-340 and SKOV3 cells was significantly inhibited by miR-340 Based on three bioinformatics algorithms (TargetScan, (Fig. 2e). In addition, flow cytometry demonstrated that miRanda, and PITA), one potential binding site of miR- cell apoptosis was markedly increased in miR-340- 340 in the FHL2 3′-UTR was predicted. To test specific overexpressing A2780 and SKOV3 cells compared with regulation through this predicted binding site, a dual- miR-NC cells (Fig. 2f). Moreover, we found that miR-340 luciferase reporter system was employed. We constructed overexpression downregulated E2F1 and pRb (Ser795), two reporter vectors consisting of the luciferase coding but enhanced the levels of p27, p21, and caspase-3 sequence followed by the 3′-UTR of FHL2 (Wt-FHL2 3′ (Fig. 2g). These results indicate that miR-340 has an UTR) or mutant 3′-UTR (Mut-FHL2 3′UTR) (Fig. 5a). inhibitory effect on OC cell proliferation in vitro. Co-transfection experiments showed that miR-340 To determine whether miR-340 affects tumor formation decreased the luciferase activity of Wt-FHL2 3′UTR, but in vivo, we performed an in vivo tumorigenesis study by had almost no effect on Mut-FHL2 3′UTR in both subcutaneously injecting Lv-miR-340-A2780 cells into HEK293T and SKOV3 cells (Fig. 5b), indicating that nude mice. The mice in the Lv-miR-340-A2780 and FHL2 is a potential target of miR-340. Furthermore, control Lv-miR-NC groups were euthanized 30 days after endogenous FHL2 mRNA and protein levels were inoculation, and the average tumor volumes and weights reduced in Lv-miR-340-A2780 and Lv-miR-340-SKOV3 were significantly reduced in Lv-miR-340-A2780 groups cells compared with the levels in their corresponding (Fig. 3a–c). By immunohistochemical analysis, Ki67 was control cells (Fig. 5c, d). Similarly, immunofluorescence markedly decreased in the Lv-miR-340 group tumors, staining showed that FHL2 levels decreased in both the while caspase-3 expression was increased in the Lv-miR- nucleus and cytoplasm of Lv-miR-340-A2780 and Lv- 340 group tumors compared with that in the Lv-miR-NC miR-340-SKOV3 cells (Fig. 5e). Consistent with this, the group tumors (Fig. 3d, e). These results demonstrate that protein levels of FHL2 were also decreased in miR-340- miR-340 can exert a significant inhibitory effect on overexpressing tumor samples from nude mice (Fig. 3a), tumorigenesis in vivo. as shown by immunohistochemistry and immunoblot analysis (Fig. 5f). Collectively, these data indicate that miR-340 inhibits migratory and invasive abilities of OC FHL2 is a novel direct target of miR-340. cells in vitro and in vivo To examine the effect of miR-340 on OC cell migration Knockdown of FHL2 mimics the effects of miR-340 in OC and invasion, Lv-miR-340-A2780 or Lv-miR-340-SKOV3 cells cells and their corresponding control Lv-miR-NC cells The observations that miR-340 inhibits OC progression were cultured in Transwell chambers pre-coated with or and that FHL2 is a direct target of miR-340 led us to without Matrigel. As shown in Fig. 4a, b, compared with hypothesize that suppression of FHL2 will have similar that of Lv-miR-NC cells, Lv-miR-340-A2780 and Lv-miR- effects on OC tumorigenesis. To test this hypothesis, 340-SKOV3 cells both showed significantly decreased siRNA targeting FHL2 were utilized to suppress its migratory and invasive ability. Consistent with this, in an expression, and knockdown efficiency was assessed by in vitro wound-healing assay, overexpression of miR-340 both western blot and real-time RT-PCR (Fig. 6a, Sup- inhibited cell migration and impeded closure of a scrat- plementary Fig. S4A). As expected, knockdown of FHL2 ched area of cells compared with that of the miR-NC cells blocked the proliferation of OC cells, as assessed by col- (Fig. 4c and Supplementary Fig. S3A). ony formation assay (Fig. 6b), arrested the cell cycle at the To examine the effect of miR-340 on OC metastasis G1-S transition (Fig. 6c, Supplementary Fig. S4B), and in vivo, Lv-miR-340 cells and their corresponding control promoted cell apoptosis in A2780 and SKOV3 cells Lv-miR-NC cells were injected intraperitoneally into nude (Fig. 6d, Supplementary Fig. S4C). Consistent with the mice. After 70 days, the mice that had been injected with effects of miR-340 overexpression, knockdown of FHL2

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Fig. 2 miR-340 inhibits growth of ovarian cancer cells and promotes apoptosis in vitro. A2780 and SKOV3 cells were transduced with miR-340- overexpressing lentivirus (Lv-miR-340) or control lentivirus (Lv-miR-NC). a, b Cell proliferation was determined at the indicated time points by MTS assay (a) and colony formation assay (b). c Cell cycle was determined by flow cytometry. d Immunofluorescence staining shows that the cell proliferation marker Ki67 was decreased in Lv-miR-340 cells compared with that in Lv-miR-NC cells. Scale bar, 50 μm. e Effect of miR-340 on cell proliferative ability of A2780 and SKOV3 cells was examined by EdU incorporation assay. Scale bar, 50 μm. f Cell apoptotic rate was increased in Lv-miR-340 cells as determined by flow cytometry of cells with Annexin V-PE/7AA-D double staining. g Levels of E2F1, pRb (Ser795), p27, p21, and caspase-3 were detected in Lv-miR-340 cells by western blot. Data are shown as means ± SD from three independent experiments. *P <0.05,**P < 0.01, ***P < 0.001 by Student’s t-tests

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Fig. 3 miR-340 suppresses ovarian tumor growth in vivo. Subcutaneous xenografts of A2780 cells infected with miR-340-overexpressing lentivirus (Lv-miR-340) or control lentivirus (Lv-miR-NC) (n = 5). a Images of the tumors at autopsy from nude mice are presented. b Tumor volumes were measured at the indicated time points. c Average weight of xenografted tumors was measured. d, e Immunohistochemical (IHC) staining of Ki67 (d) and caspase-3 (e) in xenografted tumors from Lv-miR-340-A2780 cells or control cells. Magniﬁcation ×400; scale bar, 50 μm. Data are shown as means ± SD. *P < 0.05, ***P < 0.001 by two-way ANOVA or Student’s t-tests

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Fig. 4 miR-340 inhibits migratory and invasive ability of OC cells in vitro and in vivo. a, b Stably upregulating miR-340 reduced migration (a) and invasion (b) of A2780 and SKOV3 cells in vitro. c Wound healing assay indicted that miR-340 overexpression inhibited healing ability of A2780 and SKOV3 cells. d–f Lv-miR-340 OC cells and corresponding Lv-miR-NC cells were injected intraperitoneally into BALB/c nude mice that were euthanized after 70 days (n = 4–5). d The images showing smaller ascites volumes in mice injected with Lv-miR-340 cells than in control. e Number of nodules was signiﬁcantly lower in mice injected with Lv-miR-340 cells when compared with that of the control group. f Fluorescence imaging revealed that mice injected with Lv-miR-340 cells had weaker green ﬂuorescence signals on the mesentery than did the control group. Data are shown as means ± SD from three independent experiments, unless noted otherwise. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t-tests

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Fig. 5 FHL2 is a direct target of miR-340. a Nucleotide resolution of predicted miR-340-binding site in the FHL2 mRNA 3′-UTR; mutated binding sites are in red letters. b Luciferase activities were measured in HEK293T and SKOV3 cells transfected with reporter plasmids containing wild type (Wt) or mutant type (Mut) FHL2 3′-UTR together with miR-340 mimetic or control mimetic. Relative mRNA (c) and protein (d) levels of FHL2 in Lv-miR-340- A2780 and Lv-miR-340-SKOV3 cells were measured by real-time RT-PCR and western blot. e Immunoﬂuorescence staining showed that FHL2 was decreased in Lv-miR-340 cells compared with that in Lv-miR-NC cells. Scale bar, 50 μm. f FHL2 expression was decreased in xenografted tumors from Lv-miR-340-A2780 cells by IHC staining (top, magniﬁcation ×400; scale bar, 50 μm) and western blot (bottom). Data are shown as means ± SD from three independent experiments. *P < 0.05, ***P < 0.001 by Student’s t-tests

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Fig. 6 Silencing FHL2 reduces proliferation, migration, and invasion of OC cells. A2780 and SKOV3 cells were transfected with siFHL2 or siCON. a FHL2 expression was detected by western blot. b Cell proliferation was determined by colony formation assay. c Cell cycle was determined by flow cytometry. d Cell apoptotic rate was increased in siFHL2 knockdown cells by flow cytometry of cells with Annexin V-PE/7AA-D double staining. Silencing FHL2 expression markedly reduced the ability of migration (e) and invasion (f) in OC cells. Data are shown as means ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P<0.001 by Student’s t-tests also inhibited OC cell migration and invasion (Fig. 6e, f, FHL2 significantly enhanced β-catenin reporter activity Supplementary Fig. S4D, E). (TopFlash) in A2780 and SKOV3 cells (Fig. 7a). Con- versely, miR-340 had an antagonistic effect on β-catenin miR-340-FHL2 axis regulates Wnt/β-catenin signaling in OC reporter activity (Fig. 7b). However, FHL2 and miR-340 cells had no effect on the activity of FopFlash, a negative FHL2 is known to play an oncogenic role in several control β-catenin reporter (Fig. 7a, b). These results types of cancers13, and it has been implicated in Wnt/ showed that Wnt/β-catenin signaling was negatively – β-catenin signaling17 19. Therefore, we first determined regulated by miR-340 and positively regulated by FHL2, whether FHL2 is also involved in Wnt/β-catenin signaling which offers a plausible explanation for the suppressive in OC. Indeed, we found that overexpression of effects of the miR-340-FHL2 axis in OC cells. More

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Fig. 7 miR-340-FHL2 axis regulates Wnt/β-catenin signaling in OC cells. a, b β-catenin reporter assay in A2780 or SKOV3 cells with FHL2 overexpression (a) or miR-340 overexpression (b). Data are shown as means ± SD from three independent experiments. *P < 0.05 by Student’s t-test. c Effects of miR-340 on protein levels of total β-catenin, phosphorylated β-catenin (Ser33/37/Thr41), cyclin D1, p53, PUMA, E-cadherin, and N-cadherin by western blot. d Levels of total β-catenin and phosphorylated β-catenin (Ser33/37/Thr41) in nucleus and cytoplasm of Lv-miR-340 SKOV3 cells. e FHL2 partially restored the levels of total β-catenin, phosphorylated β-catenin (Ser33/37/Thr41), cyclin D1, and p53 regulated by miR-340 interestingly, we found that FHL2 knockdown or ectopic and PUMA, was signiﬁcantly induced in FHL2- miR-340 overexpression increased the levels of phos- knockdown and miR-340-overexpressing cells (Fig. 7c phorylated β-catenin (Ser33/37/Thr41) and reduced the and Supplementary Fig. S5A). Moreover, we found that total β-catenin expression in both A2780 and SKOV3 ectopic overexpression of miR-340 increased the expres- cells. Correspondingly, a Wnt target gene, the cell cycle sion of E-cadherin and suppressed the expression of N- regulator cyclin D1, was signiﬁcantly inhibited. In addi- cadherin, which were only slightly affected by FHL2 tion, expression of the apoptosis-associated genes, p53 knockdown in both A2780 and SKOV3 cells (Fig. 7c and

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Supplementary Fig. S5A). Subcellular distribution analysis demonstrated that ectopic miR-340 significantly showed that total β-catenin expression was decreased in decreased cell proliferation, migration, and invasion, but both the nucleus and cytoplasm of Lv-miR-340-SKOV3 promoted cell apoptosis in vitro. In addition, miR-340 cells, in which the levels of phosphorylated β-catenin inhibited tumor growth and metastasis in vivo. Moreover, (Ser33/37/Thr41) were increased, compared with that of clinical sample analysis indicated that lower levels of miR- the control cells (Fig. 7d and Supplementary Fig. S5B). 340 are associated with poor OC patient outcomes. Our Furthermore, ectopic expression of FHL2 partially data indicate that miR-340 acts as a tumor suppressor in restored total β-catenin, phosphorylated β-catenin (Ser33/ OC, in accordance with previous reports of miR-340 37/Thr41) and cyclin D1 levels, which were regulated in functioning as a tumor suppressor gene in several other Lv-miR-340-SKOV3 cells (Fig. 7e). Collectively, miR-340 cancers, including breast cancer28, gastric cancer33, glio- suppressed Wnt/β-catenin signaling via targeting FHL2 in blastoma30, and non-small cell lung cancer31. These OC cells. results demonstrate that lower miR-340 levels are associated with more aggressive disease. Clinical associations of miR-340 with FHL2 expression in OC is a disease with a poor prognosis and little progress – human OC tissues has been made to improve treatment5 7. A major factor Consistent with the gene expression pattern in OC cells, for low survival has been our poor understanding of the the levels of total β-catenin, phosphorylated β-catenin initiating events that lead to OC and how the disease (Ser33/37/Thr41), cyclin D1, p53, PUMA, E-cadherin, and progresses. Accumulating evidence has demonstrated that N-cadherin were also regulated in miR-340- epigenetic aberrations are crucial in OC initiation and overexpressing tumor samples from nude mice as development45,46. Several miRNAs have been shown to be shown by immunoblot analysis (Fig. 8a). More interest- epigenetically modified by DNA methylation and histone – ingly, the highest association score between miR-340 and modifications, similar to that of protein-coding genes47 51. FHL2 was found in 481 ovarian serous cystadenocarci- For example, DNA (cytosine-5)-methyltransferase 1 noma tissues through pan-cancer analysis by CancerMi- (DNMT1)-mediated and enhancer of zeste 2 polycomb ner (Fig. 8b). Furthermore, data analysis from TCGA repressive complex 2 subunit (EZH2)-mediated methyla- revealed that FHL2 expression was significantly upregu- tion silenced the miR-200 family and promoted the pro- lated in OC tissues with lower miR-340 levels (Fig. 8c). gression of gastric cancer and glioblastoma52. miR-34a/b/ Similarly, Oncomine analysis indicated that FHL2 was c are another frequently reported epigenetically dysregu- increased in OC tissues compared with its expression in lated miRNA family, and frequent downregulation of the normal ovary tissues (Supplementary Fig. S6A). Next, we miR-34 family was shown to be involved in tumorigenesis investigated whether miR-340 expression levels were including OC53,54. Thus, it is reasonable to surmise that associated with overall survival of cancer patients by miR-340 is modulated by aberrant methylation. An ana- analyzing data from 465 ovarian patients downloaded lysis of the ~2 kb region upstream of the miR-340 locus from TCGA database. As expected, the median overall revealed three putative CpG islands, which are usually survival was substantially reduced in patients expressing unmethylated under normal cell conditions55,56.As low levels of miR-340 compared with that of patients expected, subsequent experiments confirmed that either expressing high levels of miR-340 (Fig. 8d). Furthermore, 5-Aza-dC or TSA restored the levels of miR-340 in OC three online bioinformatics algorithms (Kaplan–Meier cells, which implies that low miR-340 expression in OC plotter, Protein Atlas, and R2: Genomics Analysis and cells is regulated by epigenetic modification. Visualization Platform) were used to analyze the asso- It is well-known that cell cycle transition and EMT are ciation of overall survival of cancer patients with FHL2, intimately connected with Wnt pathway activation that – β-catenin, and cyclin D1 expression in human OC. The leads to cell growth and metastasis57 59. Activation of results showed that higher FHL2 was associated with poor Wnt signaling through mutation of pathway components, outcomes in human OC with all three bioinformatics as well as through activation of upstream signaling algorithms, but the expression of β-catenin and cyclin D1 molecules, occurs in the majority of cancers8. In this were associated with poor patient outcomes only with study, miR-340 was observed to modulate key oncogenic Protein Atlas (Fig. 8e, Supplementary Fig. S6B). genes, including FHL2, β-catenin, and cyclin D1, involved in the Wnt pathway in OC cells. This signaling cascade Discussion suppresses cell cycle transition and EMT-related genes, In this study, we found that the expression of miR-340 thus reducing cell growth and metastasis and promoting was downregulated in OC tissues compared with its OC cell apoptosis. expression in normal ovary epithelium and endometrium. FHL2 contains four and one-half highly conserved Furthermore, we provided strong evidence to support a cysteine-rich LIM homeodomains. These unique struc- tumor-suppressive role of miR-340 in OC cells. We tures enable FHL2 to interact with many different

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Fig. 8 Clinical associations of miR-340 with FHL2 expression in human OC tissues. a Levels of total β-catenin, phosphorylated β-catenin (Ser33/ 37/Thr41), cyclin D1, p53, PUMA, E-cadherin, and N-cadherin in miR-340-overexpressing tumor samples from nude mice by immunoblot analysis. b miR-340 was significantly associated with FHL2 in 481 ovarian serous cystadenocarcinoma tissues by CancerMiner. c FHL2 expression was significantly upregulated in OC tissues with lower miR-340 levels. Means ± SEM are shown. *P < 0.05 by Student’s t-test. d, e Kaplan–Meier survival curves for OC patients according to miR-340 (d) and FHL2 (e) expression levels in tumor tissues; significance was calculated using log-rank test. f Schematic diagram of roles of miR-340-FHL2 axis on Wnt/β-catenin signaling and its function in ovarian tumorigenesis

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proteins14. The role of FHL2 in cancer is particularly cadherin, N-cadherin, p53, and PUMA levels. Apparently, intriguing because it may function as an oncogenic pro- miR-340 is modulated through epigenetic modification, tein or a tumor suppressor13. Previous reports have the verification of which needs further study. This model revealed that FHL2 promotes cell proliferation in cervi- is a work-in-progress that will likely be modified in future cal20, gastric, and colon60 cancers, but inhibits myeloid studies. For example, a recent study has shown that NF- malignancies61, and neuroblastoma62. In our study, FHL2 κB1 is another target of miR-340 involved in the tumor- was observed to be a direct target of miR-340, and igenesis of OC cells67. Nevertheless, miR-340 can be silencing FHL2 mimicked the effects of miR-340 on cell considered a tumor suppressor; it has significant value as growth, apoptosis, and metastasis in OC cells. This was a prognostic indicator for OC patients and may serve as a accomplished by depression of the Wnt pathway via therapeutic target in the future. β-catenin, as well as the downstream cell cycle and EMT β Acknowledgements signals. Accordingly, -catenin and cyclin D1 were This work was supported by National Natural Sciences Foundation of China observed to be downregulated after FHL2 knockdown in (grant NSFC-81472651, 81201589). OC cells, an effect consistent with miR-340 over- Author details expression. Furthermore, FHL2 reversed the inhibitory 1 β Institute of Genomic Medicine, Wenzhou Medical University, 325000 effects of miR-340 on the expression of -catenin and Wenzhou, Zhejiang, P. R. China. 2Department of Obstetrics and Gynecology, cyclin D1. Interestingly, we also observed that down- The Second Affiliated Hospital and Yuying Children of Wenzhou Medical regulation of miR-340 in OC was associated with poor University, 325027 Wenzhou, Zhejiang, P. R. China patient outcomes. These results support that FHL2 is a Conflict of interest direct target of miR-340, which activates the Wnt sig- The authors declare that they have no conflict of interest. naling pathway, downstream cell cycle progression, and EMT signals in OC. These findings differ from those of a Publisher’s note previous report where FHL2 was shown to regulate AKT1 Springer Nature remains neutral with regard to jurisdictional claims in transcription in ovarian granulosa cell tumor progres- published maps and institutional affiliations. sion23. The detailed mechanisms of the apparent bidir- ectional effects of FHL2 remain unclear. One possibility is Supplementary Information accompanies this paper at (https://doi.org/ 10.1038/s41419-019-1604-3). that the function of FHL2 in tumorigenesis is dependent, at least in part, on its interacting partners, as well as the Received: 12 January 2019 Revised: 7 April 2019 Accepted: 19 April 2019 tissue where it is expressed. 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